Caldesmon (CaD) has been suggested as a thin filament-associated modulatory protein in smooth muscle cells. There has been an increasing body of evidence, based on both biochemical and cellular studies, suggesting that CaD indeed plays an inhibitory role in the actomyosin interaction, and therefore, during muscle contraction. In this proposal, efforts will be focused on the elucidation of the mechanism of smooth muscle CaD's modulatory action, and to test the hypothesis that CaD is necessary for smooth muscle to function normally. Specifically, we will: (1) Characterize the in vitro properties of unheated, native CaD and evaluate the validity of the many biochemical properties obtained with the heated, renatured CaD. (2) Test the functions of CaD with the use of fragments or antibodies. Recombinant fragments or antibodies of known epitopes of CaD will be used to test their effects on the biochemical properties of intact CaD, such as the ability to interact with calmodulin, actin, tropomyosin and myosin, and the inhibitory effect on the actomyosin ATPase activity, as well as in the native actomyosin filaments and isolated smooth muscle cells. (3) Determine the inhibitory function of an active CaD peptide, GS17C. We will use native thin filaments isolated from smooth muscle as an assay system to further test the hypothesis that GS17C exerts its antagonistic effect by binding to actin filaments and competing with the endogenous CaD in the smooth muscle cells. (4) Assess the functional roles of CaD phosphorylation and calcium binding proteins. The mechanism of reversing the CaD's inhibitory actions will be further studied in terms of the level and sites of phosphorylation of CaD and its interaction with Ca2+-binding proteins such as calmodulin and caltropin. (5) Finally, to test the postulated in vivo function of CaD by genetic approaches we will over-express CaD mutants that are either deficient in phosphorylation by MAP kinase or unable to interact with calmodulin, and seek correlation between the loss of functions and the contractile properties. These studies will generate information needed to assess the functional role of CaD in smooth muscle contraction, and afford a better understanding of the thin filament-based regulatory mechanism, which may be crucial for proper diagnosis and future development of therapeutic measures of many smooth muscle related diseases, such as gastrointestinal disorders, asthma, miscarriage and hypertension.